Gel-Assisted Proteome Position Integral Shift Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Caspase 3 Substrates.

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Meng Z, Saei AA, Gharibi H, Zhang X, Lyu H, Lundström SL, Végvári Á, Gaetani M, Zubarev RA

Anal. Chem. 96 (33) 13533-13541 [2024-08-20; online 2024-08-07]

Here, we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics and demonstrates its utility in studying proteolytic events in programmed cell death. With gel-assisted proteome position integral shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: (i) a negative MW shift; (ii) an elevated ratio in a pair of a semitryptic and tryptic peptide, (iii) a negative shift in the standard deviation of MW estimated for different peptides, and (iv) a negative shift in skewness of the same data. Of these proteins, 58 molecules were previously unreported caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, the GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

Chemical Proteomics [Technology development]

PubMed 39110629

DOI 10.1021/acs.analchem.4c02051

Crossref 10.1021/acs.analchem.4c02051